CN112745281A

CN112745281A - Ethylene oxide compound, preparation method thereof and application thereof in preparing RET kinase inhibitor

Info

Publication number: CN112745281A
Application number: CN202011607830.2A
Authority: CN
Inventors: 杨文普; 李延升
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-12-30
Filing date: 2020-12-30
Publication date: 2021-05-04

Abstract

The invention belongs to the field of medicines, and relates to an ethylene oxide compound, a preparation method thereof and application thereof in preparing a RET kinase inhibitor. The structural formula of the ethylene oxide compound is shown as a formula I:

pharmacological research shows that the compound prepared by the invention has good inhibitory effect on RET kinase activity, can inhibit the growth of RET abnormal tumor cells, has obvious anti-tumor activity, and can be used for preparing RET kinase inhibitors.

Description

Ethylene oxide compound, preparation method thereof and application thereof in preparing RET kinase inhibitor

Technical Field

The invention belongs to the field of medicines, and relates to an ethylene oxide compound, a preparation method thereof and application thereof in preparing RET kinase inhibitor

Background

RET (recovered reduced transfection) is a protooncogene, and has been paid much attention at home and abroad in recent two years due to the successful development of specific small molecule inhibitors. The RET gene is located on chromosome 10q 11.2 and encodes a tyrosine kinase receptor consisting of 1100 amino acids, the RET protein. The tyrosine kinase family is a family of signaling molecules involved in almost all cancer types and is also the basis for the development of cancer-targeted therapeutic drugs. Genetic mutations in members of the tyrosine kinase family are major drivers of the development of many different types of cancer. When the RET gene is mutated and activated, it may encode abnormal proteins, and the abnormal RET gene transmits abnormal signals and causes various effects on cell growth, survival, invasion, metastasis, etc. Continued aberrant signaling may cause excessive proliferation of cells, thus leading to the development and progression of tumors.

In recent years, research shows that the RET gene has high expression and/or gene rearrangement phenomena in various malignant tumors, and the expression of RET is closely related to the occurrence and development of the malignant tumors. RET gene rearrangement is observed in non-small cell lung cancer (NSCLC), papillary thyroid carcinoma, and myelomonocytic leukemia. In clinical treatment, patient resistance is more and more likely to occur, and some drugs have obvious non-targeted toxicity, and as a new treatment scheme, the research on RET kinase inhibitors has been extensively conducted since 2012 researchers found RET fusions in non-small cell lung cancer and attempted to develop small molecule inhibitors targeting RET. Multiple target inhibitors related to RET variant tumors, including Vandetib, Cabozantinib and Lenvatinib, all show a certain antitumor effect in thyroid cancer and lung cancer. However, the response rate of these multi-target inhibitors is generally less than 50%, the selectivity to RET is not high, and severe side effects such as hypertension are often caused by simultaneous inhibition of VEGFR, which limits the use of patients.

The invention innovatively synthesizes an ethylene oxide compound, has good inhibition effect on the activity of RET kinase, can inhibit the growth of RET abnormal tumor cells, has obvious anti-tumor activity, and can be used for preparing a brand new compound of the RET kinase inhibitor, and related structural reports are not seen in the prior art.

Disclosure of Invention

In view of the above problems, the present invention aims to provide a preparation method and an application of an oxirane compound, which has a good inhibitory effect on RET kinase activity and can be used for preparing RET kinase inhibitors. In order to achieve the purpose, the invention adopts the following technical scheme.

An ethylene oxide compound, which has the following structural formula I:

another objective of the present invention is to provide a synthetic route of the oxirane compound of formula I:

further, the synthesis method of each step in the synthesis route is as follows:

1) and reacting the compound II with a compound in a general formula III at high temperature in a proper solvent for about 5 hours to obtain the target compound I.

Of partial compounds¹H-NMR (400MHz) and¹³C-NMR (125MHz) was as follows:

compound 3:¹H-NMR(400MHz,CDCl₃)δ:3.15(s,4H),3.73(s,3H),3.85(d,2H),5.80(d,1H),6.72(m,1H),6.82(d,1H),7.28(m,3H),7.87(dd,1H).¹³C-NMR(125MHz,CDCl₃)δ:30.49,35.49,51.36,120.93,121.66,122.98,123.18,126.48,128.86,133.92,134.85,135.46,140.07,140.54,143.58,166.96。

compound 4:¹H-NMR(400MHz,CDCl₃)δ:3.21(s,4H),3.58(d,2H),3.71(t,1H),5.61-5.73(m,2H),6.82(d,1H),7.24-7.31(m,3H),7.92(d,1H).¹³C-NMR(125MHz,CDCl₃)δ:31.12,36.06,63.32,122.17,123.26,123.5,126.48,128.13,128.85,132.09,134.33,134.49,135.4,139.57,139.91。

compound 5:¹H-NMR(400MHz,CDCl₃)δ:3.18(s,4H),3.56(d,2H),3.88(d,2H),5.68(m,1H),5.94(m,1H),6.82(d,1H),7.26(m,2H),7.32(t,1H),7.94(d,1H).¹³C-NMR(125MHz,CDCl₃)δ:30.29,30.32,33.34,35.76,122.14,123.52,123.69,126.78,128.85,131.36,133.54,134.22,134.43,135.65,140.16,140.66。

compound 6:¹H-NMR(400MHz,CDCl₃)δ:3.01-3.11(m,2H),3.16(d,4H),3.52(d,2H),3.95-4.08(m,2H),4.23(d,1H),4.41(d,1H),6.81(d,1H),7.14(d,1H),7.26-7.31(m,2H),7.89(dd,1H).¹³C-NMR(125MHz,CDCl₃)δ:30.20,36.32,37.72,71.00,72.96,122.22,123.86,124.58,126.71,128.00,132.14,133.42,136.50,140.16,140.66。

formula I:¹H-NMR(400MHz,CDCl₃)δ:3.08(d,2H),3.20(d,4H),3.44(m,2H),3.74(m,1H),3.95(d,1H),4.00-4.06(m,1H),6.81(d,1H),7.19(d,1H),7.25-7.29(m,2H),7.83(dd,1H).¹³C-NMR(125MHz,CDCl₃)δ:31.12,38.49,45.52,56.14,72.11,121.66,122.74,123.21,126.44,128.04,131.48,133.79,135.86,140.07,140.54。

the oxirane compound has good inhibition effect on the RET kinase activity, can inhibit the growth of RET abnormal tumor cells, and has obvious anti-tumor activity. The ethylene oxide compound has positive significance in being used as a RET kinase inhibitor, and can be further studied.

The application of the medicine for preparing the RET kinase inhibitor or the pharmaceutically acceptable salt or solvate thereof in preparing the RET kinase inhibitor is particularly provides the application of an ethylene oxide compound in preparing the RET kinase inhibitor.

Compared with the prior art, the invention has the following beneficial effects:

the compound has good effect on inhibiting the activity of RET kinase. Further, it can be seen from the research results provided by the present invention that: the compound of the invention has good kinase inhibition activity, anti-tumor activity and low toxic and side effects.

In conclusion, the application of the oxirane compound in preparing the RET kinase inhibitor has good development prospect.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

Drawings

FIG. 1: the ethylene oxide compound obtained by the invention is used for measuring the RET kinase inhibition activity.

FIG. 2: the ethylene oxide compound obtained by the invention is used for measuring the proliferation inhibition activity of RET abnormal tumor cells (TT cells).

FIG. 3: the influence of the obtained ethylene oxide compound on the RET signal path.

Detailed Description

The following synthetic examples, biological test results, are used to further illustrate the invention, but are not meant to limit the invention.

Synthesis examples

Example 1 preparation of formula I

(1) Synthesis of (E) -4- (1, 2-dihydroacenaphthylene-5-yl) -2-butenoic acid methyl ester (compound 3)

(1, 2-dihydroacenaphthen-5-yl) boronic acid (compound 2) (100mmol) was dissolved in 1, 4-dioxane (12mL), to which KF (120mmol) was added, and stirred for 30 minutes. Then, Pd (OAc) is added₂(10mmol) and a solution of methyl (E) -4-bromo-2-butenoate (Compound 1) (100mmol) dissolved in 1, 4-dioxane (100mL), the reaction mixture was stirred at room temperature for 10 hours, then it was filtered through silica gel and Et was used₂O (100mL) wash. The solvent was removed by concentration under reduced pressure to give a colorless oil, which was dissolved in diethyl ether (100ml) and crystallized by stirring to give (E) -methyl 4- (1, 2-dihydroacenaphthylene-5-yl) -2-butenoate (compound 3) as a yellow solid, 21.60g, in 85.6% yield. LC-MS (ESI, pos, ion) M/z 253.12[ M + H]。

(2) Synthesis of (E) -4- (1, 2-dihydroacenaphthylene-5-yl) butyl-2-en-1-ol (Compound 4)

To a solution of (E) -4- (1, 2-dihydroacenaphthylene-5-yl) -2-butenoic acid methyl ester (compound 3) (100mmol) in anhydrous DCM (150mL) was added dropwise at 0 deg.C under nitrogen atmosphereDiisobutylaluminum hydride (200 mmol). The reaction was stirred at room temperature for 3 hours and then at 0 ℃ by addition of 10% H₂SO₄The solution (200mL) was quenched. Standing, layering, and collecting organic layer with saturated NaHCO₃Aqueous solution (80mL) and water (80 mL). With anhydrous Na₂SO₄Drying, filtration and concentration, the residue was slurried with petroleum ether for two hours, filtered and dried to give (E) -4- (1, 2-dihydroacenaphthylene-5-yl) butyl-2-en-1-ol (Compound 4) as a brown solid, 19.65g, 87.6% yield. LC-MS (ESI, pos, ion) M/z 225.12[ M + H]。

(3) Synthesis of (E) -5- (4-bromobut-2-en-1-yl) -1, 2-dihydroacenaphthylene (Compound 5)

To a solution of (E) -4- (1, 2-dihydroacenaphthylene-5-yl) butyl-2-en-1-ol (compound 4) (100mmol) in dry DCM (150mL) at 0 deg.C under nitrogen was added PPh₃(120mmol) and NBS (120 mmol). The mixture was stirred at room temperature for 2 hours, then quenched with water (100 mL). Standing, layering, and using anhydrous Na as organic phase₂SO₄Dried, filtered and concentrated. Purification by silica gel chromatography (eluent petroleum ether) gave (E) -5- (4-bromobut-2-en-1-yl) -1, 2-dihydroacenaphthylene (compound 5) as a pale yellow solid, 26.88g, 93.6% yield. LC-MS (ESI, pos, ion) M/z 287.04[ M + H]。

(4) Synthesis of 1-bromo-4- (1, 2-dihydroacenaphthylene-5-yl) butane-2, 3-diol (Compound 6)

At room temperature, adding K₂OsO₄·2H₂O(30mmol)，(DHQD)₂AQN(75mmol)，K₃FeCN₆(22.5mmol)，K₂CO₃(22.5mmol)，NaHCO₃(22.5mmol)，MeSO₂NH₂(8mmol) was added to t-BuOH/water (═ 1/1, 75 mL). The mixture was cooled to 0 ℃ and (E) -5- (4-bromobutane was added-2-en-1-yl) -1, 2-dihydroacenaphthylene (compound 5) (8 mmol). The reaction was stirred for 24 hours while maintaining the temperature, then Na was added₂SO₄(10g) And the mixture was warmed to room temperature. Ethyl acetate (80mL) was added for extraction, the layers were separated, and the aqueous layer was further extracted with ethyl acetate (2X 80 mL). The combined organic layers were passed over anhydrous Na₂SO₄Drying, filtration and concentration gave 1-bromo-4- (1, 2-dihydroacenaphthylene-5-yl) butane-2, 3-diol (compound 6) which was used in the next step without further purification. LC-MS (ESI, pos, ion) M/z 321.04[ M + H]。

(5) Preparation of 2- (1, 2-dihydroacenaphthylene-5-yl) -1- (oxiran-2-yl) ethyl-1-ol (formula I)

The crude 1-bromo-4- (1, 2-dihydroacenaphthylene-5-yl) butane-2, 3-diol (compound 6) from the previous step was dissolved in MeOH (70mL) and K was added₂CO₃(16 mmol). The reaction was stirred at room temperature for 1 hour. Water (30mL) and ethyl acetate (70mL) were added, stirred for half an hour, the layers were separated, and the aqueous layer was extracted twice with ethyl acetate (2X 80 mL). The combined organic layers were passed over anhydrous Na₂SO₄Dried, filtered and concentrated. By silica gel chromatography (eluent petroleum ether/Et)₂O-1/1) to give 2- (1, 2-dihydroacenaphthylene-5-yl) -1- (oxiran-2-yl) ethyl-1-ol (formula i) as an off-white solid, 1.45g, 75.6% yield over two steps. LC-MS (ESI, pos, ion) M/z 241.12[ M + H]。

Test example 1 measurement of RET enzyme inhibitory Activity by the Compound of the present invention

2 ul/well of 5 XT kinase (final concentration 0.5nM) was added to 384 multi-well plates, and a blank (enzyme reaction buffer) was set as a negative control and BLU-667 was set as a positive control. 4 ul/well 2.5 Xcompound gradient (final concentration of 2500nm maximum, 3 fold gradient dilution, 10 dilution points, DMSO final concentration of 0.4%) was added and incubated at room temperature for 30 min. The reaction was initiated by adding 2 ul/well of 5 Xsubstrate (final concentration 500nM) and 2 ul/well of 5 XATP (final concentration 10uM) and allowed to react at room temperature for 30 minutes. 5 ul/well TK Antibody-Cryptate and 5 ul/well 4X Steptavidin-XL665 (final concentration 62.5uM) were added and incubated at room temperature for 60 min. Envision reads the HTRF signal. Dose effect (IC) of Compounds Using GraphPad prism5 software₅₀) And (6) carrying out analysis. As shown in the figure1, the compound of the invention and the positive control BLU-667 both had significant inhibitory effects on RET enzyme activity.

Test example 2 measurement of inhibitory Activity of the Compound of the present invention on proliferation of RET abnormal tumor cells (TT cells)

The compound obtained by the present invention was assayed for the proliferation inhibitory activity against RET abnormal tumor cells (TT cells) using the MTT method. TT cells are human medullary thyroid carcinoma cell lines containing RET C634W mutation. The cells were supplemented with 10% Fetal Bovine Serum (FBS), 1x Glutamax,1x non-essentiai amino acids, 1x Pen/Strep antibodies using Ham's F-12K (Kaighn's) medium. At 37 ℃ 5% CO₂Under the conditions of (1) conventional culture and passage. Cultured cells were trypsinized and transferred to 96-well plates (5000 cells/well). After further overnight incubation, the cells were treated with the compounds obtained according to the invention at the indicated working concentrations. After 48 hours of incubation, 20uL of MTT (5mg/ml) was added dropwise to the wells. After an additional 4 hours of incubation, the mixed media in the 96-well plate was removed and then 150uL of dimethyl sulfoxide (DMSO) was added. Next, the 96-well plate was shaken for about 15 minutes, and kept at room temperature to mix the contents. The OD was then measured at a wavelength of 490 nm. Dose effect (IC) of Compounds Using GraphPad prism5 software₅₀) And (6) carrying out analysis. As shown in FIG. 2, both the compound of the present invention and the positive control BLU-667 exhibited significant inhibition of RET enzyme activity. The results are shown in FIG. 2, and show that the compound obtained by the invention has an inhibitory effect on RET abnormal tumor cell (TT cell) proliferation.

Test example 3 Effect of the Compounds of the present invention on the RET Signaling pathway

Logarithmic phase TT cells were collected and cell suspension was added to 6-well cell culture plates (1X 10)⁶Hole/bore). After overnight incubation, the compounds of the invention (0nM, 1nM, 10nM) were added and the cells were harvested after 12h of continuous incubation. Then using Whole Cell Lysate (WCL), centrifuging, adding sample buffer solution, boiling, performing SDS-PAGE electrophoresis, transferring protein onto PVDF membrane after electrophoresis, blocking with 5% BSA, incubating primary anti-RET and phosphorylated RET (pRET (Y1062)) and internal reference protein (GAPDH)) overnight, washing, and washing HPR-labeled secondary antibody at room temperatureAfter two hours of incubation, Enhanced Chemiluminiscence (ECL) detection system was detected by post-wash chemiluminescence. As shown in FIG. 3, the compounds obtained by the present invention exert their antitumor effects by inhibiting the RET signaling pathway.

The compound prepared by the invention can inhibit the growth of RET abnormal tumor cells, has obvious antitumor activity, can be used for preparing RET kinase inhibitors, and provides a new research idea for research and development of RET abnormal tumor cell medicaments.

Claims

1. An oxirane compound, the chemical structure general formula of which is shown in formula I,

2. the oxirane compound of formula I of claim 1, prepared by a process comprising:

3. the process for producing an oxirane compound according to claim 1, which comprises the steps of: and reacting the compound II with the compound in the general formula III at high temperature in a proper solvent for about 5 hours to obtain the target compound I.

4. The process of claim 3, wherein said suitable solvent in said step (1) is selected from the group consisting of methanol, ethanol, DMF, DMAC and the like.

5. Use of an oxirane compound of claim 1 in the preparation of a RET kinase inhibitor.

6. Use according to claim 5, characterized in that the compounds obtained according to the invention are used for the preparation of a medicament for the treatment of diseases mediated by RET abnormalities, preferably cancer, fibrodysplasia and genetic disorders, wherein the cancer is preferably non-small cell lung cancer, thyroid cancer, solid tumors.